Radiation nephropathy caused by yttrium 90.
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Dose-Response Relationship, Radiation↗
Biomedical subjects
M E Robbins
Publications and source records attributed to M E Robbins.
v-Ha-RaS oncogene upregulates the 92-kDa type IV collagenase (MMP-9) gene by increasing cellular superoxide production and activating NF-kappaB.
Matrix metalloproteinase 9 (MMP-9) degrades basement membrane type IV collagen and is expressed during cellular migration and invasion. Here we show that v-Ha-Ras overexpression in rat kidney epithelial cells (REC) caused upregulation of MMP-9 gene expression in part by increasing cellular oxidant levels. v-Ha-Ras mediated the production of superoxide in Ras-transfected cells, which was associated with upregulated MMP-9 gene expression. Conversely, v-Ha-Ras expression decreased steady-state levels of mRNAs from tissue inhibitor of metalloproteinase 1 (TIMP-1), an inhibitor of MMP-9; plasminogen activator inhibitor 1 (PAI-1), which indirectly activates MMP-9 by increasing plasmin levels; and collagen IV, a substrate of MMP-9 and a major component of basement membrane. Gel mobility shift assays demonstrated that Ras overexpression enhanced NF-kappaB, but not AP-1 DNA binding to motifs in the MMP-9 gene promoter. The Ras-induced increase in NF-kappaB DNA binding could be inhibited by treatment with the antioxidants N-acetyl-L-cysteine and glutathione monoester, suggesting that intracellular oxidant levels can mediate MMP-9 transcription. Our findings identify an important role for Ras in the regulation of MMP-9 expression, and suggest that increased superoxide production can upregulate MMP-9 expression and thus contribute to malignant conversion.
Animals↗
Redox modulation of the pro-fibrogenic mediator plasminogen activator inhibitor-1 following ionizing radiation.
Fibrosis is a common form of normal tissue damage after exposure to a wide variety of insults believed to involve oxidative stress. Plasminogen activator inhibitor-1 (PAI-1) is thought to play a major role in the development of progressive fibrosis via the inhibition of extracellular matrix degradation. Because radiation causes oxidative injury, which has been shown to trigger fibrogenic responses, the present study was designed to test the hypothesis that PAI-1 expression is redox-regulated after irradiation. Irradiating rat kidney tubule epithelial cells (NRK52E) with 1-20 Gy gamma-rays led to dose-dependent increases in steady-state levels of PAI-1 mRNA and immunoreactive protein within 24 and 48 h, respectively. Enhancement of intracellular soluble thiol pools after incubation with N-acetylcysteine (2.5 mM), from 3.27 +/- 0.27 nM/mg protein to 5.34 +/- 0.52 nM/mg protein in cells incubated with N-acetylcysteine 30 min before and assessed 4 h after irradiation, abolished the radiation-induced up-regulation of PAI-1. In addition, overexpression of catalase inhibited radiation-induced increases in PAI-1 expression, suggesting a mechanistic role for hydrogen peroxide (H2O2) in regulating PAI-1 expression after oxidative insult. In support of this notion, incubating NRK52E cells with H2O2 (100 microM) also led to a nearly 3-fold increase in PAI-1 gene expression. These results demonstrate that PAI-1 is redox-regulated after exposure to ionizing radiation or H2O2 and suggest that H2O2 scavenging might represent a fundamental mechanism for modulating fibrogenic disease via inhibition of the induction of profibrogenic mediators after acute or chronic oxidative stress.
Acetylcysteine↗
The role of the tubulointerstitium in radiation-induced renal fibrosis.
The functional and morphological response of the remaining hypertrophied kidney in unilaterally nephrectomized rats to single doses of 0-20 Gy X rays was investigated. Functional and histological end points were assessed serially 4-24 weeks postirradiation. Renal irradiation led to time- and dose-dependent reductions in renal function, seen in terms of a decreased glomerular filtration rate, increased blood urea nitrogen, and reduced hematocrit. These changes were accompanied by morphological changes in the glomerular, tubular and interstitial portions of the kidney. However, dose-dependent changes were observed only in terms of tubulointerstitial lesions. Significant increases in the degree of interstitial staining for collagen type III and fibronectin were observed 24 weeks postirradiation. These increases in extracellular matrix components were accompanied by a significant increase in interstitial alpha smooth muscle actin, suggesting activation of interstitial fibroblasts into myofibroblasts. There was no evidence of glomerular Tgfb after renal irradiation. A significant increase in tubular Tgfb staining was only seen 8 weeks postirradiation. In contrast, there was a shift of staining to the interstitium such that by 24 weeks postirradiation interstitial Tgfb staining was significantly greater than that seen in controls. These findings suggest that the tubule epithelial cell and the interstitial fibroblast are both active participants in the development and/or progression of radiation-induced renal fibrosis.
Animals↗
Docosahexaenoic acid synthesis from n-3 polyunsaturated fatty acids in differentiated rat brain astrocytes.
DHA, the main n-3 PUFA in the brain, is synthesized from n-3 PUFA precursors by astrocytes. To assess the potential of this process to supply DHA for the brain, we investigated whether the synthesis in astrocytes is dependent on DHA availability. Rat brain astrocytes differentiated with dibutyryl cAMP and incubated in media containing 10% fetal bovine serum synthesized DHA from alpha-linolenic acid ([1-(14)C]18:3n-3), docosapentaenoic acid ([3-(14)C]22:5n-3), tetracosapentaenoic acid ([3-(14)C]24:5n-3), and tetracosahexaenoic acid ([3-(14)C]24:6n-3). When DHA was added to media containing a 5 microM concentration of these (14)C-labeled n-3 PUFA, radiolabeled DHA synthesis was reduced but not completely suppressed even when the DHA concentration was increased to 15 microM. Radiolabeled DHA synthesis also was reduced but not completely suppressed when the astrocytes were treated with 30 microM DHA for 24 h before incubation with 5 microM [1-(14)C]18:3n-3.These findings indicate that although the DHA synthesis in astrocytes is dependent on DHA availability, some synthesis continues even when the cells have access to substantial amounts of DHA. This suggests that DHA synthesis from n-3 PUFA precursors is a constitutive process in the brain and, therefore, is likely to have an essential function.
Animals↗
Role of antioxidant enzyme expression in the selective cytotoxic response of glioma cells to gamma-linolenic acid supplementation.
We hypothesized that the cytotoxic effect of GLA observed in glioma but not normal glial cells reflects differences in GLA metabolism and/or antioxidant enzyme levels between these cells. The PUFA content of unsupplemented glioma cells was approximately 50% of that seen in unsupplemented astrocytes. Supplementation with 20 microM GLA for 24 h led to a 230 and 22% increase in glioma and astrocyte PUFA content, respectively, such that both supplemented cell types contained similar levels of PUFA. No major differences were seen in terms of GLA metabolites retained in the cells or secreted into the media following incubation with [(3)H]-GLA. No significant differences were observed in activity of MnSOD or CuZn-SOD between the cells. However, CAT and GPx activity in the glioma cells was significantly higher and lower, respectively, than observed in normal astrocytes. GLA supplementation resulted in a significant increase in CAT activity in normal astrocytes; glioma CAT activity was unchanged. No significant change was seen in the other antioxidant enzymes following GLA supplementation. These results suggest that the cytotoxic effect of GLA on glioma cells reflects both increased PUFA content and an inability to upregulate CAT.
Animals↗
Mediators and mechanisms of radiation nephropathy.
Normal tissue radiation injury occurs after sufficient irradiation, thus limiting the curative potential of x-ray therapy. In the kidney, radiation injury results in fibrosis and, ultimately, renal failure. The mediators of fibrosis in radiation nephropathy have received scant attention. Therefore, we evaluated the sequential presence of alpha smooth muscle actin (alphasma), fibrin, collagen, and TGFbeta1 in a porcine model of radiation nephropathy using 9.8 Gy single-dose local kidney irradiation. During the 24-week study, there was progressive and significant collagen accumulation in glomeruli and in interstitium. In glomeruli, this was associated with significant mesangial alphasma expression by 2 weeks after irradiation, a further rise at 4 weeks, and then a gradual fall to baseline. Glomerular fibrin deposition was significant by 4 weeks after irradiation, and remained elevated thereafter. There was little or no glomerular TGFbeta1 expression at any time point. Tubular fibrin deposition was significant at 4 weeks after irradiation but declined thereafter. There was little or no tubulo-interstitial alphasma expression at any time after irradiation. At 6 weeks after irradiation, there was a significant peak of tubular epithelial TGFbeta1 expression that declined thereafter. The early glomerular injury is evident as mesangial alphasma expression but is not proceeded by TGFbeta1 expression. There is sustained glomerular fibrin deposition with deposition of fibrin in tubular lumens, suggesting that tubular fibrin derives and flows out from injured glomerular tufts. We conclude that i) alphasma expression is an early marker of glomerular radiation injury, presaging scarring; ii) fibrin deposition is involved in glomerular and tubular radiation injury; and iii) TGFbeta1 is not an early event in radiation nephropathy, and not apparent in glomeruli in this model, but may correlate with later tubulo-interstitial fibrosis. Thus, the mediators of scarring in this model differ according to time after injury and also according to the affected tissue compartment.
Actins↗
Long-term effects of total-body irradiation on the kidney of Rhesus monkeys.
PURPOSE: To investigate the long-term effects of total-body irradiation (TBI) on kidneys in non-human primates. METHODS AND MATERIALS: The kidneys of Rhesus monkeys were histologically examined at 6-8 years after TBI with low single doses of 4.5-8.5Gy or two fractions of 5.4Gy. The kidneys of age-matched non-irradiated monkeys served as controls. Irradiation was performed on adult monkeys aged about 3 years; 6-8 years later animals were sacrificed and the kidneys removed and processed for histology. A semi-quantitative scoring system was used to evaluate overall histological damage. Glomerular changes were also morphometrically analysed according to previously published criteria. In selected dose groups (pro)thrombotic and inflammatory changes were investigated by immunostaining cryosections with antibodies against von Willebrand factor (vWF), leukocytes and macrophages. RESULTS: Histological changes were generally mild and only seen in kidneys irradiated with doses higher than 7 Gy. Glomerular changes were characterized by increased mesangial matrix and capillary dilatation. Tubulo-interstitial changes included hypercellularity, fibrosis and mild tubular atrophy. The mean glomerular area expressing vWF protein in the irradiated kidneys was not different from that in the age-matched controls. Numbers of infiltrating leukocytes were not significantly different between irradiated kidneys and controls. However, slightly increased numbers of macrophages were present in the renal cortex after irradiation. CONCLUSIONS: Renal damage after TBI of Rhesus monkeys with single doses of 4.5-8.5 Gy or two fractions of 5.4 Gy was mild, even after follow-up times of 6-8 years.
Adrenal Cortex↗
Irradiation of rat tubule epithelial cells alters the expression of gene products associated with the synthesis and degradation of extracellular matrix.
PURPOSE: To determine the ability of radiation to modulate kidney tubule epithelial cell expression of various molecules involved in regulating extracellular matrix accumulation (collagen types I and III, fibronectin, plasminogen activator-inhibitor 1 (PAI-1), TGF-beta and tissue inhibitor of metalloproteinases-2 (TIMP-2)) and degradation (plasminogen activators u-PA or t-PA, MMP-2 and MMP-9). MATERIALS AND METHODS: NRK52E rat tubule epithelial cells were placed in serum-free medium 24 h prior to irradiation with single doses of 1.0-10.0 Gy 137Cs gamma-rays. After irradiation, cells were maintained in serum-free medium for up to 48 h. RESULTS: Irradiation of NRK52E cells was associated with significant dose-dependent increases in collagen I (p<0.05) and PAI-1 (p< or =0.002) mRNA. Collagen III mRNA levels also exhibited a dose-dependent increase; however, this increase failed to reach levels that were statistically significant. Fibronectin mRNA levels were unaltered following irradiation. Radiation also led to an isoform-specific alteration in TGF-beta expression; TGF-beta1 mRNA increased, TGF-beta3 mRNA decreased. The amount of TGF-beta protein secreted into the medium was unchanged following irradiation; however, there was a fivefold increase in the relative amount of active TGF-beta. Irradiation was also associated with differential changes in MMP expression: active MMP-2 levels increased, while MMP-9 levels were unaltered; PA secretion into the medium was unchanged following irradiation. CONCLUSIONS: Irradiation of rat kidney tubule epithelial cells leads to altered production of various molecules associated with extracellular matrix accumulation and degradation.
Animals↗
A novel immunological model for the study of prostate cancer.
The Dunning R-3327 rat prostatic adenocarcinoma is a widely accepted model for in vivo experimental studies of prostate cancer. We have previously derived phenotypically distinct cell lines from a s.c. tumor resulting from the inoculation of the R-3327-5 subclone into Copenhagen rats. In this study, we report studies using a gelatin sponge model for the delivery of tumor cells and the retrieval of tumor-specific leukocytes responsive to different prostatic cell lines. S.c. preimplanted sponges were inoculated with tumor cells previously selected for differential properties of tumor formation and metastasis and examined for leukocyte content at time points of 1, 3, and 5 weeks after tumor cell inoculation. Cytospin and flow cytometric analyses revealed fewer tumor-associated leukocytes present in sponges inoculated with tumorigenic R-3327-5' and R-3327-5'B lines, with lesser sponge degradation, than in experiments with the nontumorigenic R-3327-5'A line, suggestive of a tumor cell-induced immunomodulatory mechanism. Morphological studies indicate an intermittent tumor growth pattern that gradually disappears in sponges inoculated with the nontumorigenic R-3327-5'A cells but a robust growth pattern in sponges inoculated with the tumorigenic cell lines. Cytokine analyses show the secretion of higher levels of active transforming growth factor-beta by the more invasive and metastatic lines. Total transforming growth factor-beta levels are higher in the epithelial, tumorigenic R-3327-5'B line. Additionally, the more tumorigenic lines secrete interleukin 10, a potent immunosuppressive molecule. In this report, we demonstrate the ability to retrieve viable leukocyte populations from a prostate tumor line bearing sponges, which offers an important model for further in vitro and in vivo manipulations and holds promise for testing adoptive immunotherapeutic strategies.
Adenocarcinoma↗
The selective cytotoxicity of gamma-linolenic acid (GLA) is associated with increased oxidative stress.
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Animals↗
Eicosapentaenoic acid alters manganese superoxide dismutase immunoreactive protein levels in normal but not malignant central nervous system derived cells.
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Animals↗
Angiotensin II-induced modulation of rat mesangial cell phenotype.
Inhibition of angiotensin II (AII) can ameliorate the severity of experimental radiation nephropathy. To determine the ability of AII to modulate mesangial cell phenotype, primary cultures of rat mesangial cells (passage number 6-11) were placed in serum-free medium 24 h prior to addition of AII (10(-9)-10(-5) M); control cells received serum-free medium alone. Cells were maintained in serum-free medium for a further 48 h. Addition of AII to quiescent mesangial cells resulted in significant (P < 0.05) time- and/or dose-dependent increases in Fn and Pail mRNA and/or immunoreactive protein. No significant change was observed in terms of Tgfb1 mRNA. A significant increase in total Tgfb1 protein (P < 0.01) secreted by AII-treated mesangial cells was noted; however, this increase was primarily in terms of latent TGF-beta; the relative proportion of active TGF-beta secreted decreased after AII incubation. AII had no effect on the activity of Mmp2 or Mmp9. However, AII-treated mesangial cells did show an increase in the amount of tissue inhibitor of metalloproteinase-2 (Timp2) immunoreactive protein secreted into the medium. The AII-mediated increase in Pail mRNA levels appeared due in part to activation of the AT1 receptor and was independent of TGF-beta; co-incubation with TGF-beta-neutralizing antibody failed to inhibit the AII-mediated increase in Pail mRNA. Thus mesangial cells treated with AII exhibit a pro-fibrosis phenotype.
Angiotensin II↗
Irradiation of rat mesangial cells alters the expression of gene products associated with the development of renal fibrosis.
To determine the ability of radiation to modulate mesangial cell expression of various molecules involved in promoting extracellular matrix (ECM) accumulation [fibronectin, plasminogen activator-inhibitor 1 (Pai1), and tissue inhibitor of metalloproteinase-2 (Timp2)] and degradation (Tgfb, plasminogen activators u-PA or t-PA, matrix metalloproteinases Mmp2 and Mmp9), primary cultures of rat mesangial cells (passage number 6-11) were placed in serum-free medium 24 h prior to irradiation with single doses of 0.5-20 Gy (137)Cs gamma rays. After irradiation, cells were maintained in serum-free medium for a further 48 h. Irradiation of quiescent mesangial cells resulted in significant (P < 0.05) time- and dose-dependent increases in Fn and Pai1 mRNA and/or immunoreactive protein. Despite an increase in Tgfb1 mRNA, there was little evidence for an increase in total Tgfb protein. Indeed, active levels remained unaltered after irradiation. Irradiation led to differential changes in MMP expression; active Mmp2 levels increased, while Mmp9 levels appeared unaltered. In addition, secretion of plasminogen activators into the medium was unchanged after irradiation, while secretion of Timp2 increased. We conclude that irradiating mesangial cells leads to altered production of various molecules involved in accumulation and degradation of extracellular matrix.
Animals↗
Radiation-induced alterations in rat mesangial cell Tgfb1 and Tgfb3 gene expression are not associated with altered secretion of active Tgfb isoforms.
Despite evidence of selective radiation-induced modulation of expression of rat mesangial cell Tgfb gene isoforms, it is unclear whether these changes in gene expression are accompanied by changes in protein secretion. To address this issue, primary cultures of rat mesangial cells (passage number 6- 11) were placed in serum-free medium 24 h prior to irradiation with single doses of 0.5-20 Gy of (137)Cs gamma rays. After irradiation, cells were maintained in serum-free medium for a further 24 h. Irradiation of quiescent mesangial cells resulted in a significant (P </= 0.05) dose-independent increase in steady-state levels of Tgfb1 mRNA 24 h postirradiation. In contrast, steady-state levels of Tgfb3 mRNA exhibited a dose-dependent reduction after irradiation; this reduction was statistically significant after doses of 5 and 10 Gy compared to control cells (P </= 0.05). These radiation-induced changes in Tgfb gene expression were associated with modest increases in Tgfb protein as determined using mink lung epithelial cells transfected with the Pai1 promoter-luciferase construct. Twenty-four hours after a single dose of 5 Gy, the total Tgfb protein secreted by the mesangial cells was 181 +/- 2.0% of that secreted by unirradiated control cells (P </= 0.01). However, this increase was seen in terms of latent Tgfb protein; radiation failed to increase significantly the amount of active Tgfb protein secreted by mesangial cells. Both quiescent and irradiated rat mesangial cells secreted active Tgfb as primarily the Tgfb3 isoform. These data reinforce the need to interpret changes in Tgfb gene expression with caution.
Animals↗
A morphometric analysis of glomerular and tubular alterations following fast-neutron irradiation of the pig and monkey kidney.
PURPOSE: The morphologic responses of the pig and monkey kidney to fractionated fast-neutron irradiation were assessed. METHODS AND MATERIALS: The right kidney of approximately 14-week-old female Large White pigs was irradiated with 6.6-12.2 Gy of fast neutrons (42 MeVd-->Be) given as 12 fractions over 18 days; the left kidney served as the contralateral unirradiated kidney. Both kidneys were removed at necropsy 2 years postirradiation. In addition, the remaining hypertrophied kidney of four unilaterally nephrectomized adult rhesus monkeys was irradiated with a total dose of 11.0 Gy fast neutrons (45 MeVp-->Be) given in an identical fractionation regimen to that used in the pig studies. These kidneys were removed when the animals exhibited renal failure, between 32-94 weeks postirradiation. Glomeruli were assessed for the presence of pathologic features, including intercapillary eosinophilic material (ICE), ectatic capillaries, thrombi, hemorrhage, and sclerosis. The relative proportion of renal cortex occupied by glomeruli, interstitium, normal, or abnormal tubules was determined using a Chalkley point grid. RESULTS: The incidence of normal glomeruli, ectatic capillaries, thrombosis, and periglomerular fibrosis were significantly different in the irradiated pig kidneys compared with the unirradiated contralateral kidneys (p < or = 0.02). Linear regression analysis demonstrated a significant dose relationship in terms of normal glomeruli, ectatic capillaries, and ICE (r > or = 0.64; p < or = 0.04). Irradiation was also associated with a significant (p < 0.0001) decrease and increase in the volume of renal cortex occupied by normal and abnormal tubules, respectively. Similar morphometric changes were noted in the irradiated monkey kidneys. CONCLUSIONS: The morphologic changes seen in the pig and monkey kidney after fractionated irradiation with fast neutrons are similar to those previously noted after single-dose or fractionated-photon irradiation. These findings support the hypothesis that the development of radiation nephropathy in these various models involves common pathophysiological mechanisms.
Animals↗
Pharmacologic modification of radiation-induced late normal tissue injury.
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Angiotensin Receptor Antagonists↗
Repair, repopulation and cell cycle redistribution in rat foot skin.
The influence of the phenomena of the repair of sublethal damage, repopulation and the role of the reassortment of surviving clonogenic target cells within the cell cycle have been examined in the foot skin of rats using a series of split dose experiments. The dose-related incidence of moist desquamation was used as an end-point. Initially the iso-effect dose for moist desquamation (ED50) increased with an increasing time interval (1-22 h) between two equal fractions. This effect was attributed to the well established phenomenon of the repair of sublethal damage. This appeared to be maximal with a 22 h gap between fractions. A further increase in the time interval, from 2-7 days, between two equal fractions resulted in a decrease in the ED50 value for moist desquamation. The phenomenon is most likely to be explained by a shortening of the cell cycle time in surviving epithelial target cells as repopulation first initiated. With intervals between two fractions of greater than 10 days the ED50 for moist desquamation again increased. This is likely to represent an increase in the number of epidermal target cells (repopulation). Further evidence for the effect of a reassortment of cells in the cell cycle has come from another study in which a half-tolerance priming dose of 16.8 Gy was followed by three daily fractions starting 48 or 125 h after the priming dose. The ED50 for moist desquamation based on the total fractionated dose (three fractions) was significantly lower (P < 0.05) after the longer time interval, i.e. fractions given on days 5, 6 and 7 after the primary dose. These findings were supported by the results of a cell proliferation kinetic study and jointly question the validity of a frequently made assumption of equal biological effect per fraction in a prolonged fractionated irradiation schedule.
Animals↗